Fibroin-derived protein composition

ABSTRACT

A protein composition derived from silk fibroin, which composition possesses enhanced solubility and stability in aqueous solutions. The primary amino acid sequence of native fibroin is modified in the SDP such that cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains are reduced or eliminated. Additionally, the composition can have a serine content that is reduced by greater than 40% compared to native fibroin protein, and the average molecular weight of the SDP is less than about 100 kDa.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/680,186 filed Nov. 11, 2019, which issued as U.S. Pat. No. 11,045,524on Jun. 29, 2021 and which is a continuation of U.S. patent applicationSer. No. 15/912,295 filed Mar. 5, 2018, which issued as U.S. Pat. No.10,471,128 and which is a continuation of U.S. patent application Ser.No. 15/212,086 filed Jul. 15, 2016, which issued as U.S. Pat. No.9,907,836 and which is a continuation of U.S. patent application Ser.No. 14/831,473 filed Aug. 20, 2015, which issued as U.S. Pat. No.9,394,355 and which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application Nos. 62/039,675 filed Aug. 20, 2014 and62/193,790 filed Jul. 17, 2015, which applications are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 7, 2015, isnamed 114.008US1_SL.txt and is 515 bytes in size.

BACKGROUND OF THE INVENTION

Silk fiber and secreted proteins from the domesticated silkworm Bombyxmori have been used for centuries in the textile industry. The secretedproteins have more recently been used as a biomaterial for biomedicalapplications, including as a structural component and as a proteinsolution. Natively, silkworm proteins exist as an amalgam of the silkproteins fibroin and sericin, in which sericin serves as a glue-likesubstance that binds with fibroin and maintains the shape of the cocoon.Removal of sericin, such as through detergent-mediated extraction, or inhigh-heat and high-alkaline washing, results in sericin-free fibroinfibers that include heavy and light chain fibroin proteins associatedthrough a single disulfide linkage. Conversion of these fibrils intowater-soluble silk fibroin protein requires the addition of aconcentrated heavy salt (e.g., 8-10M lithium bromide), which interfereswith inter- and intra-molecular ionic and hydrogen bonding that wouldotherwise render the fibroin protein water-insoluble.

Applications of silk fibroin proteins typically require the removal ofthe high LiBr salt concentrations, such as through the use of dialysis,so that the salts do not interfere with proper material function in agiven environment. Without these salts to compete with ionic andhydrogen bonding of the solubilized silk fibroin, silk fibroin proteinsolutions are relatively unstable, are vulnerable to proteinaggregation, and often precipitate out of aqueous solutions. Theaggregation is thought to occur through interactions between fibroinproteins, and then subsequent material gelation driven throughbeta-sheet secondary protein structure formation between the hydrophobicamino acid motifs of the fibroin heavy chains. Upon formation of thesestructures, the transition from soluble fibroin solution to insolublefibroin gel is rapid and is largely irreversible, thereby limitingapplication of the solution for aqueous solution-based applicationsbecause of limited material shelf-life.

To combat the gelling propensity of aqueous fibroin, attempts have beenmade to minimize protein aggregation and subsequent beta-sheetformation. Lowering the fibroin concentration in solution is acolligative approach aimed to attenuate the protein-proteininteractions, which precede the formation of these structures, yet mayresult in a fibroin solution that is too dilute for relevant proteinapplications. Alternatively, modifications to the aqueous solution thatwould impede protein aggregation and/or beta-sheet formation (e.g.,solution pH, addition of stabilizing excipients) may forestall theseevents. However, these modifications and chemical additions can limitdownstream applications by increasing biological toxicity or byintroducing incompatible agents in the solution. Accordingly, what isneeded is a silk-derived protein (SDP) material that is resistant toaggregation and that has a shelf-life stability profile useful acrossvarious industries.

A novel strategy to avoid the aforementioned vulnerabilities of aqueoussilk fibroin is to modify the biochemical structure and qualities of thesilk fibroin protein itself rather than the aqueous solutionenvironment. Toward this end, modifications to the silk fiber extractionprocess and/or the conditions involved in the production of aqueous silkfibroin can impact the primary sequence of amino acids, and therefore,the chemistry responsible for protein aggregation and beta-sheetformation. As such, the development of a process for modifying silkfibroin materials could dramatically extend the stability and shelf-lifeof a silk solution product.

SUMMARY

The invention provides a protein composition derived from silk fibroin.The composition intrinsically possesses enhanced solubility andstability in aqueous solutions. In one embodiment, the inventionprovides a protein composition prepared by a process comprising heatingan aqueous fibroin solution at an elevated pressure. The aqueous fibroinsolution includes lithium bromide at a concentration of at least 8M. Theaqueous fibroin solution is heated to at least about 105° C. (221° F.)under a pressure of at least about 10 PSI for at least about 20 minutes,to provide the protein composition. The polypeptides of the proteincomposition comprise less than 8.5% serine amino acid residues, and theprotein composition has an aqueous viscosity of less than 5 cP as a 10%w/w solution in water.

In other embodiments, the invention provides a protein compositionprepared by a process comprising heating an aqueous fibroin solution atan elevated pressure, wherein the aqueous fibroin solution compriseslithium bromide at a concentration of 9-10M, and wherein the aqueousfibroin solution is heated to a temperature in the range of about 115°C. (239° F.) to about 125° C. (257° F.), under a pressure of about 14PSI to about 20 PSI for at least about 20 minutes; to provide theprotein composition. The protein composition can include less than 6.5%serine amino acid residues and the protein composition can have anaqueous viscosity of less than 10 cP as a 15% w/w solution in water.

The invention also provides a fibroin-derived protein composition thatpossesses enhanced stability in aqueous solution, wherein: the primaryamino acid sequences of the fibroin-derived protein composition differsfrom native fibroin by at least by at least 4% with respect to thecombined difference in serine, glycine, and alanine content; cysteinedisulfide bonds between the fibroin heavy and fibroin light proteinchains are reduced or eliminated; the composition has a serine contentthat is reduced by greater than 25% compared to native fibroin protein;and wherein the average molecular weight of the silk derived protein isless than about 100 kDa.

In another embodiment, the invention provides a fibroin-derived proteincomposition that possesses enhanced stability in aqueous solution,wherein: the primary amino acid sequences of the fibroin-derived proteincomposition differs from native fibroin by at least by at least 6% withrespect to the combined difference in serine, glycine, and alaninecontent; cysteine disulfide bonds between the fibroin heavy and fibroinlight protein chains are reduced or eliminated; the composition has aserine content that is reduced by greater than 40% compared to nativefibroin protein; and wherein the average molecular weight of the silkderived protein is less than about 96 kDa.

The invention further provides a fibroin-derived protein compositionthat possesses enhanced stability in aqueous solutions, wherein: theprimary amino acid sequences of the fibroin-derived protein compositionis modified from native silk fibroin; cysteine disulfide bonds betweenthe fibroin heavy and fibroin light protein chains are reduced oreliminated; the average molecular weight of the silk derived protein isless than about 100 kDa; and the fibroin-derived protein compositionmaintains an optical absorbance at 550 nm of less than 0.25 for at leasttwo hours after five seconds of ultrasonication.

In another embodiment, the invention provides a fibroin-derived proteincomposition that possesses enhanced stability in aqueous solutions,wherein: the primary amino acid sequences of the fibroin-derived proteincomposition is modified from native silk fibroin such that they differfrom native fibroin by at least by at least 5% with respect to thecombined difference in serine, glycine, and alanine content; cysteinedisulfide bonds between the fibroin heavy and fibroin light proteinchains are reduced or eliminated; the average molecular weight of thesilk derived protein is less than about 96 kDa; and the fibroin-derivedprotein composition maintains an optical absorbance at 550 nm of lessthan 0.2 for at least two hours after five seconds of ultrasonication.

The fibroin-derived protein composition can be isolated and/or purifiedas a dry powder or film, for example, by dialysis and/or filtration.Alternatively, the fibroin-derived protein composition can be isolatedand/or purified as a stable aqueous solution, which can be modified foruse as a food or beverage composition, or as a therapeutic formulation,such as an ophthalmic formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1. Flowchart illustrating key processing steps for the generationof both SDP solution and prior art silk fibroin solution. The SDPProduction Process contains an additional step (italicized in center) toenhance solution stability over time, which is not performed during theprior art silk fibroin solution production process.

FIG. 2. Picture showing results of the Lawrence Stability Test for astable SDP solution (Sample 1, on left, produced by the processdescribed in Example 1), and a prior art silk fibroin solution (Sample2, on right, produced by standard hydrolysis conditions). Visualinspection reveals that Sample 1 is a stable aqueous solution that hasnot gelled, while Sample 2 has gelled, and therefore is not a stableaqueous solution.

FIG. 3. Picture of a gel showing process-mediated modification ofaqueous silk fibroin protein to SDP solution. The picture shows themolecular weight (MW) distribution of an SDP Solution (Lane 3,autoclaved) versus a prior art silk fibroin solution (Lane 2,non-autoclaved). A protein standard ladder (Lane 1) and associatedweights (numbers to the left of Lane 1) are provided as a reference ofMW. A prominent MW band at 23-26 kDa in Lane 2 is noteworthy and isentirely absent following the autoclaving process, indicating thatdegradation of the fibroin light chain contributes to the enhancedstability of the SDP protein material. Also, a clear shift is observedin MW range of fibroin protein following autoclaving (Lane 3),indicating modification of the natural silk fibroin protein to the SDPmaterial composition.

FIG. 4A-B. Images demonstrating that (A) SDP Solution material does notgel, while (B) Prior Art Silk Fibroin solution material gelled within 2hours following ultrasonication.

FIG. 5. Impact of the fibroin processing as described herein on proteinsolution stability and viscosity. Summary graph illustrating solutionviscosity as a function of protein concentration in Prior Art SilkFibroin (PASF), PASF heated to 225° F. for 30 minutes (PASF-225° F.),and SDP. PASF and PASF-225° F. demonstrated a sharp increase inviscosity in solutions of >75 mg/g (7.5% w/w) and could not beconcentrated higher than 200 mg/g without proteins falling out ofsolution. In contrast, SDP maintained a low viscosity throughout allconcentrations, and was able to be concentrated to levels exceeding 240mg/g.

FIG. 6A-B. Heat treatment of silk fibroin protein generates pyruvate,indicating modification of the fibroin primary structure. (A) Summarygraph showing pyruvate concentrations in silk fibroin protein solutions(50 mg/mL) exposed to no heat, or upon heating to 65° C. (˜150° F.), 90°C. (˜200° F.), and 99° C. (˜210° F.). (B) The duration of heat treatmentfurther enhances pyruvate formation from silk fibroin. A 30-minuteexposure of aqueous silk proteins to 99° C. (˜210° F.) causes a nearlytwofold increase in pyruvate levels relative to pyruvate levels at thetime this temperature was initially achieved, and over fourfold that ofnon-heated samples.

FIG. 7A-B. Heat treatment alters the amino acid composition of nativesilk protein. (A) Summary graph showing serine composition as apercentage of total amino acids in fibroin protein in non-heated (i.e.,‘no heat’/prior art) silk protein solution (left column, 10% serine) andSDP solution previously subject to processing as described in Example 1(e.g., ˜121° C., 17 psi) for 30 minutes (‘heat’; right column, 5.7%serine). Heat treatment reduced serine composition by over 40% in SDPsolution samples when compared to prior art silk fibroin solutionsamples. (B) Summary graph depicting percent concentrations of glycineand alanine in a prior art silk fibroin protein solution (left ‘no heat’columns) and a heat and pressure processed (˜121° C., 17 psi) SDPsolution (right ‘heat’ columns). Heat and pressure processingfacilitates an increase in levels of glycine and alanine relative toprior art silk fibroin solution controls.

FIG. 8. Various sample formulations were placed on a hydrophobic waxsurface: phosphate buffered saline (PBS), TheraTears (TT), Blink,Systane Balance (SB), and a 5% w/v SDP formulation (shown on the leftside of the figure). Formulation solution spreading was imaged, and thespreading area was then measured at time points before and aftermechanical spreading (data shown on the right side of the figure). Aftermechanical spreading, the SDP formulation showed significantly enhancedspreading (by over threefold) compared to all other sample formulations.

FIG. 9A-C. Amino acid transformation in SDP impairs secondary proteinstructures and permits dissolution. (A) FTIR spectra of bothpre-processed and post-processed water-annealed samples of PASF Solutionand SDP Solution. The prior art samples show significant beta-sheetsignature peaks post water-annealing around 1624 cm⁻¹ and 1510 cm⁻¹,while the spectrum of the SDP solution does not indicate formation ofbeta-sheet peaks and instead indicates significantly reduced beta-sheetcontent post-processing. (B) Representative images of SDP and PASF silksolutions desiccated to form films. (C) Subsequent dissolution of SDPfilms in water was complete, indicating that no beta-sheet secondarystructures had formed; however, PASF films were unable to dissolveentirely, rendering a mixture of partially dissolved PASF andundissolved beta-sheet-containing protein aggregates.

FIG. 10. Enzymatic cleavage of silk fibroin with trypsin enhancesinstability and accelerates gel formation but does not affect SDP.Summary graph depicting absorbance (550 nm) of ultrasonicated PASFpreviously treated with trypsin for indicated time points. Increasingabsorbance indicates fibroin beta-sheet formation which culminates ingel formation, demonstrating solution instability.

FIG. 11. Summary graph depicting longitudinal optical absorbance (550nm) of PASF solutions treated with 0 (control), 10, or 100 mMdithiothreitol (DTT) 30 minutes prior to ultrasonication. The dataprovide an indicator of secondary structure formation in PASF or SDPsolutions over time. Accumulating beta-sheet formation, shown as anincreasing absorbance, occurs immediately after sonication of PASFsolutions (e.g., within 30 minutes and subsequently increasing).Conversely, SDP exhibits no tendency towards secondary structureformation. Reduction of disulfide bridges with DTT retards beta-sheetand subsequent gel formation compared to control PASF, but the materialultimately forms significant amounts of beta-sheets. In contrast, SDPshowed no tendency to form secondary structures and remained stable.Thus, reduction of disulfide bridges in native fibroin improvesstability but does not prevent gel formation, and longitudinalinstability of PASF is abolished in SDP.

FIG. 12. Heating PASF in the absence of lithium bromide (LiBr) impairsgelation. Summary graph depicting optical absorbance (550 nm) from PASFsolutions not heated (control) or heated at −200° F. for indicateddurations prior to ultrasonication. Solution heating caused an increasein basal absorbance which increased with heating duration relative tonon-heated PASF. All PASF solutions demonstrated an increase inabsorbance over time, indicating change in protein properties. Incontrast, SDP exhibited no change in absorbance followingultrasonication throughout the duration of the experiment.

DETAILED DESCRIPTION

The invention provides a protein composition derived from silk fibroin.The protein composition possesses enhanced solubility and stability inaqueous solutions. The primary amino acid sequence of native fibroin ismodified in the fibroin-derived protein composition such that cysteinedisulfide bonds between the fibroin heavy and fibroin light proteinchains are reduced or eliminated. Additionally, the composition can havea serine content that is reduced by greater than 40% compared to nativefibroin protein, and the average molecular weight of the proteins in thecomposition is less than about 100 kDa.

Definitions

The following definitions are included to provide a clear and consistentunderstanding of the specification and claims. As used herein, therecited terms have the following meanings. All other terms and phrasesused in this specification have their ordinary meanings as one of skillin the art would understand. Such ordinary meanings may be obtained byreference to technical dictionaries, such as Hawley's Condensed ChemicalDictionary 14^(th) Edition, by R. J. Lewis, John Wiley & Sons, New York,N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a component” includes a plurality of such components, so that acomponent X includes a plurality of components X. It is further notedthat the claims may be drafted to exclude an optional element. As such,this statement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” “other than”, and thelike, in connection with any element described herein, and/or therecitation of claim elements or use of “negative” limitations.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrases “one or more” and “at least one” are readily understood by oneof skill in the art, particularly when read in context of its usage. Forexample, the phrase can mean one, two, three, four, five, six, ten, 100,or any upper limit approximately 10, 100, or 1000 times higher than arecited lower limit.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percentages, proximate to the recited range that are equivalentin terms of the functionality of the individual ingredient, element, thecomposition, or the embodiment. The term about can also modify theendpoints of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percentages or carbon groups) includes each specific value,integer, decimal, or identity within the range. Any listed range can beeasily recognized as sufficiently describing and enabling the same rangebeing broken down into at least equal halves, thirds, quarters, fifths,or tenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, for use in an explicit negative limitation. The term“contacting” refers to the act of touching, making contact, or ofbringing to immediate or close proximity, including at the cellular ormolecular level, for example, to bring about a physiological reaction, achemical reaction, or a physical change, e.g., in a solution, in areaction mixture, in vitro, or in vivo.

For a therapeutic application, an “effective amount” refers to an amounteffective to treat a disease, disorder, and/or condition, or to bringabout a recited effect. For example, an effective amount can be anamount effective to reduce the progression or severity of the conditionor symptoms being treated. Determination of a therapeutically effectiveamount is within the capacity of persons skilled in the art. The term“effective amount” is intended to include an amount of a compositiondescribed herein, or an amount of a combination of peptides describedherein, e.g., that is effective to treat or prevent a disease ordisorder, or to treat the symptoms of the disease or disorder, in ahost. Thus, an “effective amount” generally means an amount thatprovides the desired effect.

For process and preparation applications, an “effective amount” refersto an amount effective to bring about a recited effect, such as anamount necessary to form products in a reaction mixture. Determinationof an effective amount is typically within the capacity of personsskilled in the art, especially in light of the detailed disclosureprovided herein. The term “effective amount” is intended to include anamount of a compound or reagent described herein, or an amount of acombination of compounds or reagents described herein, or conditionsrelated to a process described herein, e.g., that is effective to formthe desired products in a reaction mixture. Thus, an “effective amount”generally means an amount that provides the recited desired effect.

Fibroin is derived from the Bombyx mori silkworm cocoon. The proteinfibroin includes a heavy chain that is about 350-400 kDa in molecularweight and a light chain that is about 25 kDa in molecular weight,wherein the heavy and light chains are linked together by a disulfidebond. The primary sequences of the heavy and light chains are known inthe art. The fibroin protein chains possess hydrophilic N and C terminaldomains, and alternating blocks of hydrophobic/hydrophilic amino acidsequences allowing for a mixture of steric and electrostaticinteractions with surrounding molecules in solution. At lowconcentration dilutions (1% or less) the fibroin protein molecule isknown to take on an extended protein chain form and not immediatelyaggregate in solution. The fibroin protein is highly miscible withhydrating molecules such as HA, PEG, glycerin, and CMC, has been foundto be highly biocompatible, and integrates or degrades naturally withinthe body through enzymatic action. Native fibroin, or prior art silkfibroin (PASF), is known in the art and has been described by, forexample, Daithankar et al. (Indian J. Biotechnol. 2005, 4, 115-121).

The terms “silk-derived protein” (SDP) and “fibroin-derived protein” areused interchangeably herein. These materials are prepared by theprocesses described herein involving heat, pressure, and a highconcentration of a heavy salt solution. Therefore ‘silk-derived’ and‘fibroin-derived’ refers to the starting material of the process thatmodifies the silk fibroin protein to arrive at a protein compositionwith the structural, chemical and physical properties described herein.

Fibroin-Derived Protein Composition Preparation

The fibroin-derived protein composition described herein possessesenhanced stability compared to native fibroin in aqueous solutions. Theenhanced stability achieved by the fibroin-derived protein composition,also referred herein as a silk-derived protein (SDP), allows thematerial to remain in solution significantly longer than thenative/prior art silk fibroin proteins (referred to herein as PASF). Theenhanced stability of the SDP material also allows for the preparationof SDP solutions of high concentration without aggregation,precipitation, or gelation. In commercial applications such as withfood, beverage, eye drops, or applications requiring protein to besoluble in solution, the enhanced stability provides suitably lengthyshelf-life and increased quality of the product by reducing proteinaggregation. Potential aggregation of protein in solution negativelyimpacts a product's desired performance for a particular application.The ability to concentrate the SDP to high constitutions in solution(over 50% w/v or >500 mg/mL) is significantly advantageous forinventorying a useful working solution that can be used as-is or dilutedfor any number of applications. Examples of such applications are theuse of SDP as an ingredient in food, beverage, or ophthalmicformulations as a protein supplement or additive.

The enhanced stability in aqueous solutions is derived from transformingthe primary amino acid sequences of the native fibroin protein into theSDP material. The changes in the primary sequence decreases thesusceptibility of the molecules to aggregate. Aggregation eventuallyleads to gel formation. In the transformation of the native fibroin,both serine and cysteine amino acids are cleaved in the presence of highheat and dehydrating conditions.

Similarly, Patchornik et al. (J. Am. Chem. Soc. 1964, 86, 1206)demonstrated that a dehydroalanine (DHA) intermediate is formed fromserine and cysteine in solution. The amino acid degradation is furtherdriven when in the presence of a strong dehydrating solvent system, suchas the 50-55% w/v LiBr solution as described herein, in which a hydrideshift takes place to induce removal of water. The degradation reactioncan take place in the presence of hydroxide ions (e.g., pH 7.5 to pH11), which further drives cleavage of the DHA intermediate. Thiscleavage forms an amide, a pyruvoyl peptide, and LiBr. One viablechemical mechanism is outlined in Scheme 1 for a serine amino acid,which scheme is also applicable for cysteine amino acids. Chemicalalteration of the serine and cysteine amino acids of the PASF proteininto a DHA intermediate with further hydrolytic cleavage leads toenhanced solution stability of the SDP products.

The cleavage reaction discussed above significantly affectmacromolecular properties of the resulting peptides, which results in anaqueous solution of stabilized SDP material. The initial proteinaggregation of fibroin is believed to be instigated by interactions ofthe native fibroin heavy and light chains at the cysteine amino acids asdescribed by Greying et al. (Biomacromolecules 2012, 13(3): 676-682).The cysteine amino acids within the fibroin light and heavy proteinchains interact with one another through disulfide linkages. Thesedisulfide bridges participate in fibroin protein aggregation and gelnetwork flocculation. Without the native fibroin light chain present,the proteins are significantly less susceptible to aggregation.Therefore, the process described herein effectively reduces the nativefibroin light chain's ability to form disulfide bonds by reducingcysteine content and thus reducing or eliminating disulfide bond-formingcapability. Through this mechanism, the transformative process describedherein functionally stabilizes the resulting SDP in solution by reducingor eliminating the ability to form cysteine-derived aggregations.

In addition to aggregation-inducing disulfide bridges, thesusceptibility of the silk fibroin to further aggregate into flocculatedstructure is also driven by the protein's amino acid chemistry asdescribed by Mayen et al. (Biophysical Chemistry 2015, 197:10-17).Molecular modeling of silk fibroin serine, alanine, and glycine aminoacid sequences have shown that the presence of serine enhances initialprotein-to-protein interaction through a greater propensity to createhydrogen bonding between adjacent fibroin protein chain moieties. Themodels demonstrate that reduced serine and increased alanine and glycinedecrease the initial propensity for protein aggregation. The molecularmodeling observations indicate that by altering the native amino acidchemistry of the fibroin protein a material could be generated thatwould have higher stability in aqueous solution.

One strategy to accomplish enhanced stability is to eliminate chargedfunctional groups, such as hydroxyls, from the protein. Due to therelatively high electronegativity of hydroxyl groups, this chemistry candrive both hydrogen bonding with available hydrogen atoms andnon-specific charge interactions with positively charged amino acidgroups. Almost 12% of the native fibroin protein's content is composedof serine, which bears a hydroxyl functional group. Therefore, byreducing the availability of hydroxyl groups that facilitate hydrogenbonding, the overall protein stability in solution may be enhanced. Theprocess described herein effectively reduces the amount of serinecontent and increases the relative alanine and glycine content, whicheliminates the number of available hydroxyl groups available to createhydrogen bonds. Through this mechanism the process described hereinfunctionally stabilizes the resulting SDP in solution for extendedperiods of time (e.g., at least several [6-8] months, and/or for morethan 1.5 years; extended studies are ongoing, indicating that stabilitymay be maintained for more than 2 years, or more than 3 years).

In addition to the reduction of cysteine and serine moieties, solventcharge interaction is important for stabilizing a protein solution.After initial protein flocculation, the gelation process is believed tocontinue to drive closer associations among the native fibroin heavychains, which leads to both intra- and inter-molecular beta-sheetformation among hydrophobic blocks of the heavy chains. Once significantbeta-sheet formation occurs, the fibroin solution transitions to a gel.As the solution transitions to a gel, and the fibroin becomes insolubleand is no longer useful as a solution-based product. To preventgelation, the pH of the SDP solution can be raised to high alkalinity toenhance stability, for example over a pH of 7.5. As a result, theincreased pH produces additional free hydroxyl groups that form avalence shield around the SDP molecules in solution. The formed valenceshield acts to produce a zeta potential that stabilizes the protein byreducing protein-protein interactions derived from hydrogen bonding ornon-specific charged and/or hydrophobic interactions. Thefibroin-transformation process functionally stabilizes processed SDP insolution through this mechanism and others.

SDP material can be prepared by the following process.

-   -   1. Silk cocoons are prepared by removing pupae material and        pre-rinsing in warm water.    -   2. Native fibroin protein fibers are extracted from the gum-like        sericin proteins by washing the cocoons in water at high water        temperature, typically 95° C. or more, at an alkaline pH.    -   3. The extracted fibroin fibers are dried and then dissolved        using a solvent system that neutralizes hydrogen bonding between        the beta-sheets; a 54% LiBr aqueous solution of 20% w/v silk        fibroin protein is effective for this neutralization step.    -   4. The dissolved fibroin protein in LiBr solution is processed        in an autoclave environment (˜121° C. [˜250° F.], at ˜15-17 PSI        pressure, for approximately 30 minutes at temperature).    -   5. The heat processed fibroin protein and LiBr solution are then        dialyzed to remove lithium and bromide ions from solution. At        this point in the process the material has been chemically        transformed to SDP.    -   6. The dialyzed SDP is then filtered to remove any non-dissolved        aggregates and contaminating bioburden.

The SDP solution is produced using a distinctly different process thanthe process used for current silk fibroin solution production, asschematically illustrated in FIG. 1. Notably, the autoclaving of thesilk fibroin protein while it is combined with LiBr in solutioninitiates chemical transitions to produce the stabilized SDP material.The fibroin protein is dissolved in LiBr solution, which neutralizeshydrogen bonding and electrostatic interactions of the solubilizednative fibroin protein. This leaves the protein without specificsecondary structure confirmations in solution. As a result, thethermodynamic energy required to hydrolyze covalent bonding within thefibroin protein chain is at its lowest energy requirements to initiatehydrolytic cleavage.

In one embodiment the temperature is set to 121° C. for 30 minutes at15-17 PSI autoclave conditions. However, in various embodiments, theprocessing conditions may be modified to stabilize the SDP material tovarying degrees. In other embodiments, additional protein solubilizationagents can be used in the process, including other or additional halidesalts such as calcium chloride and sodium thiocyanate, organic agentssuch as urea, guanidine hydrochloride, and1,1,1,3,3,3-hexafluoroisopropanol, additional strong ionic liquidsolution additives such as calcium nitrate and1-butyl-3-methylimidazolium chloride, or a combination thereof.

Fibroin-Derived Protein Compositions

The invention provides a protein composition derived from silk fibroin,which composition possesses enhanced solubility and stability in aqueoussolutions. In one embodiment, the invention provides a proteincomposition prepared by a process comprising heating an aqueous fibroinsolution at an elevated pressure. The aqueous fibroin solution includeslithium bromide at a concentration of at least 8M. The aqueous fibroinsolution is heated to at least about 105° C. (221° F.) under a pressureof at least about 10 PSI for at least about 20 minutes, to provide theprotein composition. The polypeptides of the protein compositioncomprise less than 8.5% serine amino acid residues, and the proteincomposition has an aqueous viscosity of less than 5 cP as a 10% w/wsolution in water.

In other embodiments, the invention provides a protein compositionprepared by a process comprising heating an aqueous fibroin solution atan elevated pressure, wherein the aqueous fibroin solution compriseslithium bromide at a concentration of 9-10M, and wherein the aqueousfibroin solution is heated to a temperature in the range of about 115°C. (239° F.) to about 125° C. (257° F.), under a pressure of about 15PSI to about 20 PSI for at least about 20 minutes; to provide theprotein composition. The protein composition can include less than 6.5%serine amino acid residues and the protein composition can have anaqueous viscosity of less than 10 cP as a 15% w/w solution in water.

The invention also provides a fibroin-derived protein composition thatpossesses enhanced stability in aqueous solution, wherein: the primaryamino acid sequences of the fibroin-derived protein composition differsfrom native fibroin by at least by at least 4% with respect to thecombined difference in serine, glycine, and alanine content(fibroin-derived vs. PASF); cysteine disulfide bonds between the fibroinheavy and fibroin light protein chains are reduced or eliminated; andthe composition has a serine content that is reduced by greater than 25%compared to native fibroin protein. The average molecular weight of thefibroin-derived protein composition can be less than about 100 kDa andgreater than about 25 kDa.

In another embodiment, the invention provides a fibroin-derived proteincomposition that possesses enhanced stability in aqueous solution,wherein: the primary amino acid sequences of the fibroin-derived proteincomposition differs from native fibroin by at least by at least 6% withrespect to the combined difference in serine, glycine, and alaninecontent; cysteine disulfide bonds between the fibroin heavy and fibroinlight protein chains are reduced or eliminated; and the composition hasa serine content that is reduced by greater than 40% compared to nativefibroin protein. The average molecular weight of the fibroin-derivedprotein composition can be less than about 96 kDa and greater than about25 kDa.

The invention further provides a fibroin-derived protein compositionthat possesses enhanced stability in aqueous solutions, wherein: theprimary amino acid sequences of the fibroin-derived protein compositionis modified from native silk fibroin; cysteine disulfide bonds betweenthe fibroin heavy and fibroin light protein chains are reduced oreliminated; the average molecular weight of the fibroin-derived proteincomposition is less than about 100 kDa and greater than about 25 kDa;and the fibroin-derived protein composition maintains an opticalabsorbance at 550 nm of less than 0.25 for at least two hours after fiveseconds of ultrasonication. For example, a 5% w/w solution of theprotein composition can maintain an optical absorbance of less than 0.1at 550 nm after five seconds of ultrasonication at 10 Hz and 20%amplitude, which are the standard conditions used for ultrasonicationdescribed herein.

In another embodiment, the invention provides a fibroin-derived proteincomposition that possesses enhanced stability in aqueous solutions,wherein: the primary amino acid sequences of the fibroin-derived proteincomposition is modified from native silk fibroin such that they differfrom native fibroin by at least by at least 5% with respect to thecombined difference in serine, glycine, and alanine content; cysteinedisulfide bonds between the fibroin heavy and fibroin light proteinchains are reduced or eliminated; the average molecular weight of thefibroin-derived protein composition is less than about 96 kDa andgreater than about 25 kDa; and the fibroin-derived protein compositionmaintains an optical absorbance at 550 nm of less than 0.2 for at leasttwo hours after five seconds of ultrasonication.

In various embodiments, the fibroin-derived protein composition can beisolated and/or purified as a dry powder or film, for example, bydialysis and/or filtration. Alternatively, the fibroin-derived proteincomposition can be isolated and/or purified as a stable aqueoussolution, which can be modified for use as a food or beveragecomposition, or as a therapeutic formulation, such as an ophthalmicformulation. The invention therefore also provides a food or beveragecomposition that includes a protein composition described herein and afood or beverage component. Food components can include one or more ofsimple sugars, disaccharides, carbohydrates, fats, oils, vitamins,minerals, and water. Beverage components can include one or more ofwater, a coloring agent (e.g., a synthetic colorant, or a naturalcolorant such as saffron), vitamins, and minerals.

In various embodiments, the amino acid composition of thefibroin-derived protein differs from the amino acid composition ofnative fibroin by at least by at least 4%, by at least 4.5%, by at least5%, or by at least 5.5%, or by at least 6%, with respect to the contentof serine, glycine, and alanine combined.

The composition can have a serine content that is reduced by greaterthan 25%, by greater than 30%, by greater than 35%, by greater than 40%,or by greater than 45%, compared to the serine content of native fibroinprotein.

The average molecular weight of the fibroin-derived protein compositioncan be less than about 100 kDa, less than about 98 kDa, less than about96 kDa, less than about 95 kDa, less than about 90 kDa, less than about85 kDa, less than about 80 kDa, less than about 75 kDa, or less thanabout 70 kDa. In various embodiments, the average molecular weight ofthe fibroin-derived protein composition can be greater than about 30kDa, greater than about 35 kDa, greater than about 40 kDa, greater thanabout 50 kDa, greater than about 60 kDa, or greater than about 70 kDa.Accordingly, the (weight average) average molecular weight of thefibroin-derived protein composition can be about 30 kDa to about 100kDa, about 30 kDa to about 96 kDa, about 30 kDa to about 90 kDa, about35 kDa to about 80 kDa, about 35 kDa to about 70 kDa, about 40 kDa toabout 60 kDa. In various embodiments, the average molecular weight ofthe fibroin-derived protein composition is about 60 kDa to about 80 kDa,about 50 kDa to about 70 kDa, about 40 kDa to about 60 kDa, about 30 kDato about 50 kDa, about 35 kDa to about 45 kDa, or about 40 kDa to about43 kDa.

In various embodiments, the protein composition has an aqueous viscosityof less than 4 cP as a 10% w/w solution in water. In additionalembodiments, the protein composition has an aqueous viscosity of lessthan 10 cP as a 24% w/w solution in water.

In some embodiments, the protein composition is soluble in water at 40%w/w without any precipitation observable by ocular inspection.

In various embodiments, the protein composition does not gel uponultrasonication of an aqueous solution of the protein composition atconcentrations of up to 10% w/w. In additional embodiments, the proteincomposition does not gel upon ultrasonication of an aqueous solution ofthe protein composition at concentrations of up to 15% w/w, up to 20%w/w, up to 25% w/w, up to 30% w/w, up to 35% w/w, or up to 40% w/w.

In some embodiments, the protein composition comprises less than 8%serine amino acid residues. In other embodiments, the proteincomposition comprises less than 7.5% serine amino acid residues, lessthan 7% serine amino acid residues, less than 6.5% serine amino acidresidues, or less than 6% serine amino acid residues.

In some embodiments, the protein composition comprises greater than46.5% glycine amino acids, relative to the total amino acid content ofthe protein composition. In other embodiments, the protein compositioncomprises greater than 47% glycine amino acids, greater than 47.5%glycine amino acids, or greater than 48% glycine amino acids.

In some embodiments, the protein composition comprises greater than 30%alanine amino acids, relative to the total amino acid content of theprotein composition. In other embodiments, the protein compositioncomprises greater than 30.5% alanine, greater than 31% alanine, orgreater than 31.5% alanine.

In some embodiments, the protein composition completely re-dissolvesafter being dried to a thin film. In various embodiments, the proteincomposition lacks beta-sheet protein structure in aqueous solution. Incertain embodiments, the protein composition maintains an opticalabsorbance in aqueous solution of less than 0.25 at 550 nm after atleast five seconds of ultrasonication.

In some embodiments, protein composition is in combination with water.The protein composition can completely dissolve in water at aconcentration of 10% w/w, or even greater concentrations such as 15%w/w, 20% w/w, 25% w/w, 30% w/w, 35% w/w, or 40% w/w. In someembodiments, the protein composition can be isolated and purified, forexample, by dialysis, filtration, or a combination thereof.

In various embodiments, the protein composition enhances the spreadingof an aqueous solution comprising the protein composition and ophthalmicformulation components, for example, compared to the spreading of acorresponding composition that does not include the protein composition.The enhanced spreading can result in an increase in surface area of theaqueous solution by greater than twofold, or greater than threefold.

In some embodiments, the protein composition does not form a gel atconcentrations up to 20% w/v, up to 30% w/v, or up to 40% w/v. Theprotein composition can remain in solution up to a viscosity of at least9.8 cP.

In some embodiments, the fibroin-derived protein composition can haveglycine-alanine-glycine-alanine (GAGA) (SEQ ID NO: 1) segments of aminoacids that comprise at least about 47.5% of the amino acids of thefibroin-derived protein composition. The fibroin-derived proteincomposition can also have glycine-alanine-glycine-alanine (GAGA) (SEQ IDNO: 1) segments of amino acids that comprise at least about 48%, atleast about 48.5%, at least about 49%, at least about 49.5%, or at leastabout 50%, of the amino acids of the protein composition. In variousembodiments, the fibroin-derived protein composition can haveglycine-alanine (GA) segments of amino acids that comprise at leastabout 59% of the amino acids of the fibroin-derived protein composition.The fibroin-derived protein composition can also have glycine-alanine(GA) segments of amino acids that comprise at least about 59.5%, atleast about 60%, at least about 60.5%, at least about 61%, or at leastabout 61.5%, of the amino acids of the protein composition.

In another embodiment, the primary amino acid sequences of thefibroin-derived protein composition differs from native fibroin by atleast by at least 6% with respect to the combined difference in serine,glycine, and alanine content; the average molecular weight of thefibroin-derived protein composition is less than about 100 kDa; and thefibroin-derived protein composition maintains an optical absorbance at550 nm of less than 0.25 for at least two hours after five seconds ofultrasonication. Thus, in one specific embodiment, the inventionprovides a fibroin-derived protein composition that possesses enhancedstability in aqueous solution, wherein: the primary amino acid sequencesof the fibroin-derived protein composition differs from native fibroinby at least by at least 6% with respect to the combined difference inserine, glycine, and alanine content; cysteine disulfide bonds betweenthe fibroin heavy and fibroin light protein chains are reduced oreliminated; the composition has a serine content that is reduced bygreater than 40% compared to native fibroin protein; and wherein theaverage molecular weight of the fibroin-derived protein composition isless than about 96 kDa.

In another embodiment, the invention provides a fibroin-derived proteincomposition that possesses enhanced stability in aqueous solutions,wherein: the primary amino acid sequences of the fibroin-derived proteincomposition is modified from native silk fibroin; cysteine disulfidebonds between the fibroin heavy and fibroin light protein chains arereduced or eliminated; the average molecular weight of thefibroin-derived protein composition is less than about 100 kDa; and thefibroin-derived protein composition maintains an optical absorbance at550 nm of less than 0.25 for at least two hours after five seconds ofultrasonication. In one specific embodiment, the primary amino acidsequences of the fibroin-derived protein composition is modified fromnative silk fibroin such that they differ from native fibroin by atleast by at least 5% with respect to the combined difference in serine,glycine, and alanine content; the average molecular weight of thefibroin-derived protein composition is less than about 96 kDa; and thefibroin-derived protein composition maintains an optical absorbance at550 nm of less than 0.2 for at least two hours after five seconds ofultrasonication.

Thus, in one specific embodiment, the invention provides afibroin-derived protein composition that possesses enhanced stability inaqueous solutions, wherein: the primary amino acid sequences of thefibroin-derived protein composition is modified from native silk fibroinsuch that they differ from native fibroin by at least by at least 5%with respect to the combined difference in serine, glycine, and alaninecontent; cysteine disulfide bonds between the fibroin heavy and fibroinlight protein chains are reduced or eliminated; the average molecularweight of the fibroin-derived protein composition is less than about 96kDa; and the fibroin-derived protein composition maintains an opticalabsorbance at 550 nm of less than 0.2 for at least two hours after fiveseconds of ultrasonication.

The invention also provides a protein composition prepared by a processcomprising heating an aqueous fibroin solution at an elevated pressure,wherein the aqueous fibroin solution comprises lithium bromide at aconcentration of at least 8M, and wherein the aqueous fibroin solutionis heated to at least about 105° C. (221° F.) under a pressure of atleast about 10 PSI for at least about 20 minutes; to provide the proteincomposition, wherein the protein composition comprises less than 8.5%serine amino acid residues and the protein composition has an aqueousviscosity of less than 5 cP as a 10% w/w solution in water. Therefore,the invention provides a method of preparing a fibroin-derived proteincomposition comprising one or more of the process steps describedherein.

In one embodiment, the concentration of lithium bromide is about 8.5M toabout 11M. In some embodiments, the concentration of lithium bromide isabout 9M to about 10M, or about 9.5M to about 10M.

In some embodiments, the aqueous fibroin solution that contains lithiumbromide is heated to at least about 107° C. (225° F.), at least about110° C. (230° F.), at least about 113° C. (235° F.), at least about 115°C. (239° F.), or at least about 120° C. (248° F.).

In some embodiments, the aqueous fibroin solution that contains lithiumbromide is heated under a pressure of at least about 12 PSI, at leastabout 14 PSI, at least about 15 PSI, or at least about 16 PSI, up toabout 18 PSI, or up to about 20 PSI.

In some embodiments, the aqueous fibroin solution that contains lithiumbromide is heated for at least about 20 minutes, at least about 30minutes, at least about 45 minutes, or at least about 1 hour, up toseveral (e.g., 12-24) hours.

In some embodiments, the protein composition has an aqueous viscosity ofless than 4 cP as a 10% w/w solution in water. In various embodiments,the protein composition has an aqueous viscosity of less than 10 cP as a24% w/w solution in water.

In some embodiments, the protein composition can be dissolved in waterat 40% w/w without observable precipitation.

In some embodiments, the fibroin has been separated from sericin.

In some embodiments, lithium bromide has been removed from the proteincomposition to provide a purified protein composition. In variousembodiments, the protein composition has been isolated and purified, forexample, by dialysis, filtration, or a combination thereof.

In various embodiments, the protein composition does not gel uponultrasonication of an aqueous solution of the composition atconcentrations of up to 10% w/w, up to 15% w/w, up to 20% w/w, up to 25%w/w, up to 30% w/w, up to 35% w/w, or up to 40% w/w.

In additional embodiments, the protein composition has properties asdescribed above, and amino acid compositions as described aboveregarding serine, glycine, and alanine content.

In various embodiments, the protein composition re-dissolves afterdrying as a thin film. The protein composition can lack beta-sheetprotein structure in solution. The protein composition can maintain anoptical absorbance in solution of less than 0.25 at 550 nm after atleast five seconds of ultrasonication.

In one specific embodiment, the invention provides a protein compositionprepared by a process comprising heating an aqueous fibroin solution atan elevated pressure, wherein the aqueous fibroin solution compriseslithium bromide at a concentration of 9-10M, and wherein the aqueousfibroin solution is heated to a temperature in the range of about 115°C. (239° F.) to about 125° C. (257° F.), under a pressure of about 15PSI to about 20 PSI for at least about 30 minutes; to provide theprotein composition, wherein the protein composition comprises less than6.5% serine amino acid residues and the protein composition has anaqueous viscosity of less than 10 cP as a 15% w/w solution in water.

Further embodiments of the invention are described herein below.

The invention provides a novel silk-derived protein (SDP) compositionthat is chemically distinct from native silk fibroin protein. The SDPhas enhanced stability in aqueous solution. The SDP can be used in amethod for forming a food composition, a beverage, or an ophthalmicformulation comprising combining food, beverage, or ophthalmicingredients with a protein composition described herein, for example, aprotein composition aqueous solution. The solution can include about0.01% to about 92% w/v SDP. The solution can be about 8% to about 99.9%w/v water.

In one embodiment, the SDP material with enhanced solution stability canbe used as an ingredient in a beverage for human or animal consumption,such as an ingredient or additive in a sports drink, nutrient drink,soft drink, or in bottled water. In another embodiment, the SDP materialwith enhanced solution stability can be used as an ingredient in a foodproduct, such as in dairy products, cereal, or processed foods. In yetanother embodiment, the SDP material with enhanced solution stabilitycan be used as an ingredient in an eye drop formulation, such as inartificial tears, ocular lubricants, lid scrubs, or therapeuticformulations.

In one aspect, the invention provides a process that induces hydrolysis,amino acid degradation, or a combination thereof, of fibroin proteinsuch that the average molecular weight of the protein is reduced fromabout 100-200 kDa for silk fibroin produced using prior art methods toabout 30-90 kDa, or about 30-50 kDa, for the SDP material describedherein. The resulting polypeptides can be a random assortment ofpeptides of various molecular weights averaging to the ranges recitedherein. In addition, the amino acid chemistry can be altered by reducingcysteine content to non-detectable levels by standard assay procedures.For example, the serine content can be reduced by over 50% from thelevels found in the native fibroin, which can result in increases ofoverall alanine and glycine content by 5% (relative amino acid content),as determined by standard assay procedures. The process can provide aprotein composition where the fibroin light chain protein is notdiscernable after processing, as well when the sample is run usingstandard SDS-PAGE electrophoresis methods. Furthermore, the resultingSDP material forms minimal to no beta-sheet protein secondary structurepost-processing, while silk fibroin solution produced using prior artmethods forms significant amounts of beta-sheet secondary structure. Inone embodiment, the SDP material can be prepared by processing silkfibroin fibers under autoclave or autoclave-like conditions (i.e.,approximately 120° C. and 14-18 PSI) in the presence of a 40-60% w/vlithium bromide (LiBr) solution.

In some embodiments, the invention provides a food or beverage productthat includes the SDP as an ingredient. The SDP can serve to provideadditional protein content, resulting in improved nutritional value,health benefits, and/or therapeutic advantages to the human or animalthat consumes the food or beverage. In one embodiment, the SDP isincluded in a beverage such as water, a sport drink, an energy drink, ora carbonated drink. In another embodiment, the SDP is included in foodproducts such as yogurt, energy bars, cereal, bread, or pasta.

The food or beverage product can include an effective amount of SDP,such as about 0.01% by weight to about 92% by weight of SDP. In variousembodiments, the SDP can be present in about 0.1% by weight to about 30%by weight, about 0.5% by weight to about 20% by weight, or about 1% byweight to about 10% by weight. In certain specific embodiments, the SDPcan be derived from Bombyx mori silkworm fibroin.

In another embodiment, the invention provides an ophthalmic compositionfor the treatment of dry eye syndrome in a human or mammal. Thecomposition can be an aqueous solution that includes an amount of SDPeffective for treating dry eye syndrome. For example, the aqueoussolution can include about 0.01% by weight to about 80% by weight SDP.In other embodiments, the aqueous solution can include SDP at about 0.1%by weight to about 10% by weight, or about 0.5% by weight to about 2% byweight. In certain specific embodiments, the ophthalmic composition caninclude about 0.05% w/v SDP, about 0.1% w/v SDP, about 0.2% w/v SDP,about 0.25% w/v SDP, about 0.5% w/v SDP, about 0.75% w/v SDP, about 1%w/v SDP, about 1.5% w/v SDP, about 2% w/v SDP, about 2.5% w/v SDP, about5% w/v SDP, about 8% w/v SDP, or about 10% w/v SDP. The SDP can bederived from Bombyx mori silkworm fibroin.

In various embodiments, the ophthalmic formulation can includeadditional components in the aqueous solution, such as a demulcentagent, a buffering agent, and/or a stabilizing agent. The demulcentagent can be, for example, hyaluronic acid (HA), hydroxyethyl cellulose,hydroxypropyl methylcellulose, dextran, gelatin, a polyol, carboxymethylcellulose (CMC), polyethylene glycol, propylene glycol (PG),hypromellose, glycerin, polysorbate 80, polyvinyl alcohol, or povidone.The demulcent agent can be present, for example, at about 0.01% byweight to about 10% by weight, or at about 0.2% by weight to about 2% byweight. In one specific embodiment, the demulcent agent is HA. Invarious embodiments, the HA can be present at about 0.2% by weight ofthe formulation.

The buffering or stabilizing agent of an ophthalmic formulation can bephosphate buffered saline, borate buffered saline, citrate buffersaline, sodium chloride, calcium chloride, magnesium chloride, potassiumchloride, sodium bicarbonate, zinc chloride, hydrochloric acid, sodiumhydroxide, edetate disodium, or a combination thereof.

An ophthalmic formulation can further include an effective amount of anantimicrobial preservative. The antimicrobial preservative can be, forexample, sodium perborate, polyquaterium-1 (e.g., Polyquad®preservative), benzalkonium (BAK) chloride, sodium chlorite,brimonidine, brimonidine purite, polexitonium, or a combination thereof.

An ophthalmic formulation can also include an effective amount of avasoconstrictor, an anti-histamine, or a combination thereof. Thevasoconstrictor or antihistamine can be naphazoline hydrochloride,ephedrine hydrochloride, phenylephrine hydrochloride, tetrahydrozolinehydrochloride, pheniramine maleate, or a combination thereof.

In one embodiment, an ophthalmic formulation can include an effectiveamount of fibroin-derived protein as described herein in combinationwith water and one or more ophthalmic components. The ophthalmiccomponents can be, for example, a) polyvinyl alcohol; b) PEG-400 andhyaluronic acid; c) PEG-400 and propylene glycol, d) CMC and glycerin;e) propylene glycol and glycerin; f) glycerin, hypromellose, andPEG-400; or a combination of any one or more of the precedingcomponents. The ophthalmic formulation can include one or more inactiveingredients such as HP-guar, borate, calcium chloride, magnesiumchloride, potassium chloride, zinc chloride, and the like. Theophthalmic formulation can also include one or more ophthalmicpreservatives such as sodium chlorite (Purite® preservative (NaClO₂),polyquad, BAK, EDTA, sorbic acid, benzyl alcohol, and the like.Ophthalmic components, inactive ingredients, and preservatives can beincluded at about 0.1% to about 5% w/v, such as about 0.25%, 0.3%, 0.4%,0.5%, 1%, 2%, 2.5%, or 5%, or a range in between any two of theaforementioned values.

Accordingly, the invention provides a silk derived protein (SDP)composition that possesses enhanced stability in aqueous solutions inwhich the primary amino acid sequence of native fibroin is modified fromnative silk fibroin, wherein cysteine disulfide bonds between thefibroin heavy and fibroin light protein chains reduced or eliminated;wherein the composition has a serine content that is reduced by greaterthan 40% compared to native fibroin protein; and wherein the averagemolecular weight of the silk derived protein is less than about 96 kDa.

The invention also provides an ophthalmic formulation for the treatmentof ophthalmic disorders in a human or mammal, wherein the ophthalmicformulation comprises water and an effective amount of the SDP asdescribed above. The invention further provides an ophthalmiccomposition for use as an eye treatment in a human or mammal, whereinthe ophthalmic composition comprises water, one or more of a bufferingagent and stabilizing agent, and an effective amount of the SDP asdescribed above.

The SDP is highly stable in water, where shelf life solution stabilityis more than twice that of native silk fibroin in solution. For example,the SDP is highly stable in water, where shelf life solution stabilityis more than 10 times greater compared to native silk fibroin insolution. The SDP material, when in an aqueous solution, does not gelupon sonication of the solution at a 5% (50 mg/mL) concentration. Inother embodiments, the SDP material, when in an aqueous solution, doesnot gel upon sonication of the solution at a 10% (100 mg/mL)concentration.

The SDP material can have the fibroin light chain over 50% removed whencompared to native silk fibroin protein. The SDP material can have aserine amino acid content of less than about 8% relative amino acidcontent, or a serine amino acid content of less than about 6% relativeamino acid content.

The SDP material can have a glycine amino acid content above about46.5%. The SDP material can have an alanine amino acid content aboveabout 30% or above about 30.5%. The SDP material can have no detectablecysteine amino acid content, for example, as determined by HPLC analysisof the hydrolyzed polypeptide of the protein composition.

The SDP material can form 90% less, 95% less, or 98% less beta-sheetsecondary protein structures as compared to native silk fibroin protein,for example, as determined by the FTIR analysis described in Example 8below.

The invention additionally provides an ophthalmic composition for use asan eye treatment in a human or mammal, the composition comprising anaqueous solution including an effective amount of SDP material asdescribed above, and a buffering or stabilizing agent.

The invention yet further provides an ophthalmic formulation for thetreatment of ophthalmic disorders in human or mammal, the compositioncomprising an aqueous solution including an effective amount of SDPmaterial with enhanced stability as described herein.

The invention also provides a method for forming a beverage mixture, afood composition, or an ophthalmic composition, with silk proteincomprising combining a food, beverage, or ophthalmic components with thefibroin-derived protein composition described herein.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

Example 1. Silk-Derived Protein Preparation and the Lawrence StabilityTest

Materials. Silkworm cocoons were obtained from Tajima Shoji Co., Ltd.,Japan. Lithium bromide (LiBr) was obtained from FMC Lithium, Inc., NC.An autoclave was obtained from Tuttnauer Ltd., NY. The 3,500 Damolecular-weight cutoff (MWCO) dialysis membranes were obtained fromThermoScientific, Inc., MA. An Oakton Bromide (BP) double-junctionion-selective electrode was obtained from ISE, Oakton Instruments, IL.

Processing. Two samples, SDP and prior art silk fibroin, were preparedas illustrated in FIG. 1. Briefly, SDP was produced by submergingpupae-free, cut silkworm cocoons (3-5 cuts/cocoon) into 95° C. heated,deionized water (diH₂O) containing 0.3 wt % NaCO₃ at 233 mL water/gramof cocoons. Cocoons were agitated in this solution for 75 minutes todissolve sericin, thereby separating it from the silk fibers. Thecocoons were subsequently washed four times in like dilutions of diH₂Ofor 20 minutes per rinse to remove residual sericin from the washed silkfibers. The fibers were then dried in a convection oven at 60° C. for 2hours, weighed, and dissolved in 54 wt % LiBr in water at a ratio of 4×LiBr volume per gram of extracted fiber. This solution was covered andthen warmed in a convection oven at 60° C. for 2 hours to expediteextracted fiber dissolution. The solution was then placed in anautoclave and exposed to sterilization conditions (121° C., 17 PSI,90-100% humidity) for 30 minutes to facilitate fibroin transformation.The resulting solution was allowed to cool to room temperature, thendiluted to 5% fibroin with diH₂O and dialyzed to remove LiBr salts usinga 3,500 Da MWCO membrane. Multiple exchanges were performed in diH₂Ountil Br⁻ ions were less than 1-ppm as determined in the hydrolyzedfibroin solution read on an Oakton Bromide (Br⁻) double-junctionion-selective electrode. The solution was then further filtered using a1-5 1 μm porosity filter followed by filtration through a 0.2 μmpolishing filter. This product is referred to as ‘SDP Solution’ in FIG.2.

A ‘control’ silk fibroin solution was prepared as illustrated in FIG. 1to provide the ‘Prior Art Silk Fibroin Solution’ shown in FIG. 2. Exceptthe autoclave step, the same process was performed as described above. Asampling volume (5 mL) from each sample was placed in separate 20 mLglass beakers and the beakers were sealed with foil. The samples werethen subjected to the Lawrence Stability Test.

The Lawrence Stability Test is performed by placing the aqueous proteintest solution (5% w/v, 50 mg/mL) within the autoclave chamber. Theautoclave is then activated for a cycle at 121° C., 17 PSI, for 30minutes, at 97-100% humidity. After completion of the cycle, thesolution is allowed to cool and is then removed from the autoclavechamber. The solution is then shaken to observe solution gelationbehavior. If the solution has gelled upon shaking for ˜10 seconds, thesample fails the Lawrence Stability Test. Failing the test indicatesthat the material is inherently unstable as a protein solution.

The Lawrence Stability Test was performed on both the SDP Solution andthe Prior Art Silk Fibroin Solution. The Prior Art Silk Fibroin Solutionsample gelled immediately and therefore failed the Lawrence StabilityTest. Conversely, the SDP Solution sample remained in solutionindefinitely and therefore passed the Lawrence Stability Test. The lackof gelation can be attributed to the fact that SDP Solution productionincorporated the autoclave-processing step as indicated in FIG. 1 above.An image of the different test results (not-gelled vs. gelled) is shownin FIG. 2.

Example 2. Silk-Derived Protein Molecular Weight Characterization

To evaluate the effect of processing on the molecular weightdistribution of solubilized protein, SDP Solution and Prior Art SilkFibroin Solution were subjected to polyacrylamide gel electrophoresis(PAGE), which separates proteins by molecular weight. Specifically, 15μg of each sample was mixed with running buffer containing sodiumdodecyl sulfate and dithiothreitol (Biorad Inc., CA) to remove anysecondary folding structures and disulfide bonds, respectively. Themixtures were then heated to 70° C. for 5 minutes. The mixtures wereloaded along with a 2.5-200 kDa molecular weight ladder (LifeTechnologies, CA) onto pre-cast, 4-12% polyacrylamide gradient gelscontaining Bis-Tris buffer salts (Life Technologies, CA), and thenexposed to 120V electric field for 90 minutes on a BioRad PowerPac Powersupply (BioRad Inc., CA). The gels were then removed and placed inCoomassie Blue stain for 12 hours to stain proteins, followed by 6 hoursof washing in diH₂O. The gels were then scanned on a Biorad GS-800Calibrated Desitometer (BioRad Inc., CA).

The resulting gel is shown in FIG. 3. The results show that theprocessing employed to prepare the SDP solution significantly shifts theaverage molecular weight from 150-200 kDa to less than 80 kDa (FIG. 3).The shift in molecular weight clearly indicates a transformation of theprimary and/or secondary structure of the original native fibroin. Inaddition, the fibroin light chain of fibroin is not present in the SDPafter the autoclaving process (visible at 23-26 kDa in Lane 2 for theprior art fibroin), which indicates that the fibroin light chain portionof the protein has been degraded or removed by the processing. Theseresults demonstrate that the autoclave processing transforms the nativefibroin protein to a new material that has smaller peptide fragmentsthan the native fibroin protein. The process further degrades/modifiesthe fibroin light chain. These transformations result in an SDP materialthat possesses enhanced solution stability as a result of these chemicalchanges.

Example 3. Silk-Derived Protein Stability Study

To further determine the functional impact of the autoclave process onthe stability of the resulting SDP compared to the stability of priorart fibroin, the samples were analyzed using the methods of Wang et al.(Biomaterials 2008, 29(8):1054-1064) to mimic a well-characterized modelof silk fibroin protein gelation. Volumes of both samples (0.5 mL, SDPand PASF) were added to 1.7 mL clear centrifuge tubes and were subjectedto ultrasonication (20% amplitude, 10 Hz, 15 seconds). The clear tubescontaining the solutions were then visually monitored for gel formationas a screen for gelation.

The SDP Solution samples failed to form gels, as shown in FIG. 4A. Even3 months post-sonication, the SDP samples remained in solution andlacked protein aggregation as determined by visual inspection. The PriorArt Silk Fibroin Solution sample gelled rapidly (within 2 hours)following sonication. The resulting gelled Prior Art Silk Fibroin isshown in FIG. 4B. These results further indicate that the autoclaveprocess transforms the prior art fibroin to a new material and inducesstability to the resulting SDP material.

Example 4. Impact of Heating on the Viscosity Profile of Aqueous SilkSolutions

The physicochemical properties of PASF and SDP were investigated, withparticular attention paid to the impact of protein concentration onsolution viscosity. It has been shown by Zafar et al. (Biomacromolecules2015, 16(2):606-614) that silk fibroin heavy and light chain proteinsare distinct in their rheological properties, and therefore,differential degradation rates of these constituents in PASF would imbueunpredictable changes to the viscosity of a given solution over time.Furthermore, the impact of total fibroin protein concentration onviscosity is non-linear, also shown by Zafar et al., and escalatesrapidly as purified fibroin solutions exceed 100 mg/mL, thus restrictingthe useable concentration of the protein solution for a particularapplication.

To determine whether these limitations could be overcome through aminoacid transformations that culminate in SDP, 80-100 mL of PASF orautoclave-treated SDP were generated at 50-80 mg/mL. To assess theimpact of heating PASF to a level below autoclaving conditions, PASF washeated to 225° F. for 30 minutes in a jacketed reaction vessel asdescribed above. Purified solutions were placed in 140 mm shallowplastic weigh boats in a laminar flow hood (Baker Sterilgard 56400,Class II) at ˜22° C. to facilitate evaporation. At periodic intervals,concentrating samples were collected to measure protein content(calculated in % w/w) and assess viscosity using a viscometer(Brookfield LVDV-E, spindle 00). Measurements were made at spindlerevolutions per minute (about 1-100 rpm) that permitted a torque rangethat would permit accurate viscosity measurements, measured at 25° C.,on 16 mL sample volumes.

As summarized in FIG. 5, the viscosity of PASF rose precipitously whensolutions exceeded 75 mg/g. Furthermore, PASF could not be concentratedto >200 mg/g, at which point fibroin protein began to become insoluble.PASF solutions heated to 225° F. prior to dialysis demonstrated theimpact of heat on solution viscosity. In particular, heated PASFexhibited decreased viscosities at any given concentration relative tonon-heated PASF. In contrast, SDP exhibited minimal changes in solutionviscosity at concentrations at or below 140 mg/g (FIG. 5). Furthermore,SDP viscosity remained below ˜10 cP at protein concentrations where PASFcould no longer stay in solution (e.g., at 240 mg/g). Importantly, SDPwas capable of remaining homogeneous at concentrations exceeding 400mg/g, where viscosities stayed below 150 cP.

An aqueous solution of SDP thus exhibits lower viscosity when comparedto PASF at all concentrations above 4% w/w. Additionally, gelationbegins to occur at about 20% w/w for the PASF solution at which pointaccurate viscosity measurements where not possible, while the SDPmaterial increased in concentration without exhibiting gelation,aggregating behavior, or significant increases in viscosity through 25%w/w solutions.

Taken together, these results clearly demonstrate that theprocess-related protein transformations described herein for thepreparation of SDP are needed for the production of a highlyconcentrated, low viscosity protein solution.

Example 5. Formation of Pyruvoyl Peptides

The chemical reaction illustrated in Scheme 1 described above results inthe production of a pyruvoyl peptide. The pyruvoyl peptide degrades intopyruvate, which readily detectable by a standard pyruvate assay. Todemonstrate that the application of heat and pressure (e.g., theenvironment in an autoclave) could facilitate pyruvoyl generation insilk fibroin protein processing, aqueous silk fibroin solution (5% w/vin water) was produced using the prior art method described above. Thematerial was then heated in a thermally jacketed beaker (ChemGlass, NJ)at defined temperatures up to 210° F., or just below the boiling pointof the protein solution. Specifically, Prior Art Silk Fibroin proteinsolutions were heated to ˜65° C. (˜150° F.), ˜90° C. (˜200° F.), or ˜99°C. (˜210° F.), and then sampled upon reaching these temperatures tomeasure pyruvate concentrations via colorimetric assay (Pyruvate AssayKit, MAK071, Sigma-Aldrich).

The production of pyruvate increased in both 90° C. and 99° C. heatedsamples. Pyruvate increased by 50% at 99° C. (FIG. 6A). To determinewhether pyruvate conversion was further enhanced over time, the sampleswere heated to 99° C. and maintained at this temperature for 30 minutes(FIG. 6B). Sustained heating caused a robust increase in pyruvateformation, generating more than a fourfold increase in pyruvate relativeto non-heated samples. These results indicate that upon heating the silkfibroin protein, there is a chemical conversion to pyruvoyl containingmaterial as detected by pyruvate assay. From this data it can beconcluded that within a more extreme heating environment, such as in anautoclave process, the silk fibroin protein will be stimulated toproduce pyruvate to an even greater extent. This provides furtherevidence that the final SDP product is a chemically distinct entity fromthe Prior Art Silk Fibroin.

Example 6. Amino Acid Profile Analysis

The impact of heating silk fibroin fibers dissolved in 9.3M LiBrsolution on amino acid profile was investigated using ion-exchangechromatography (AOAC Official Method 994.12, Amino Acids.com, MN).Samples were produced using the processes described in Example 1 forboth the SDP Solution and the Prior Art Silk Fibroin Solution. Thesolutions were then submitted in like-concentrations for evaluation bychromatography. Particular attention was paid to the amino acids serine,glycine, and alanine, given their prominent constitution in the silkfibroin protein primary sequence and their key roles in secondarystructure formation.

SDP solution samples were found to contain 40% less serine relative toPrior Art Silk Fibroin Solution samples (FIG. 7A), and a correspondingincrease in the levels of glycine and alanine (FIG. 7B). These resultsindicate a significant change in amino acid content, which changesresult in different (and enhanced) chemical and physical properties as aresult of the autoclave process. These results also corroborate withfindings in the literature by Mayen et al. (Biophysical Chemistry 2015.197:10-17) where increased serine content was shown to increase initialaggregation of silk fibroin proteins, while silk fibroin protein withincreasing glycine and alanine content required greater energythresholds to initially aggregate. Therefore, by reducing serine andincreasing both alanine and glycine content, the propensity (and/orpossibility) for aggregation is reduced or eliminated, leading to thegreater solution stability of SDP.

Example 7. Artificial Tear Formulations

The SDP solution was used to formulate an artificial tear for use intreating ophthalmic conditions and disorders. The artificial tears canbe specifically formulated and used for the treatment of the disorder‘dry eye’. The artificial tears can also be formulated and used fortreatment of an ocular wound created by either accidental or surgicalinsult.

Incorporation of SDP into artificial tear formulations is especiallyadvantageous because it increases the spreadability of the formulation.SDP-containing artificial tears also have an extremely long shelf-lifedue to their solution stability. The block co-polymer arrangement ofhydrophilic and hydrophobic amino acid groups located in the backbone ofthe SDP protein allows the molecules to interact with both water-solubleand water-insoluble chemistries within the tear film. When included asan ingredient in an artificial tear eye drop formulation, the SDPingredient acts to enhance the spreadability of the artificial tear,which provides additional comfort to the patient and prolonged efficacyto the product. The aggregating groups of prior art silk fibroinsolution are not required to enable this spreading property, so it isadvantageous to remove these regions to enhance protein productstability in solution over time.

The enhanced spreading ability of the artificial tear was demonstratedby comparing leading brand artificial tear products with an artificialtear formulated using the SDP ingredient. A test protocol was used toevaluate the effect of mechanical spreading on the wetting ability ofvarious eye drop products. Phosphate buffered saline (PBS), TheraTears®(TT) artificial tears by AVR, Blink® Tears eye drops by AMO, SystaneBalance® (SB) eye drops by Alcon, and a formulation containing SDP werecompared in the experiment. For reference, PBS contained 100 mmol PBSsalts in water, TT contains 0.25% wt. carboxymethyl cellulose (CMC) asthe active ingredient with additional buffering salts in water, Blinkcontains 0.2% wt. hyaluronic acid (HA) as the active ingredient withbuffering salts in water, SB contains 0.6% wt. propylene glycol (PG) asthe active ingredient with HP-guar and mineral oil as enhancingexcipients with buffering salts, and the SDP solution contained 0.25%wt. CMC with 1% wt./vol. SDP and buffering salts (i.e., 0.01M phosphatebuffer containing 137 mM NaCl).

This group of compared products included multiple demulcents andadditional active ingredient-enhancing excipients. FIG. 8 shows thatinclusion of the SDP ingredient enhances mechanical spreading ability bynearly fourfold over the leading brand formulations. This post-spreadingenhancement can be compared to the eyelid wiping across the ocularsurface. Thus, the SDP ingredient allows for better comfort to thepatient while enhancing efficacy and stability of the product.Furthermore, the addition of SDP over prior art silk fibroin solution asan ingredient is advantageous because the prior art silk fibroinsolution would gel and aggregate during the product shelf life. Theseaggregates would be unacceptable in an ophthalmic formulation based oncurrent United States Pharmacopeia (USP) requirements (ParticulateMatter in Injections: USP <788-789>).

Example 8. Analysis of Protein Secondary Structure

The impact of autoclave processing on secondary structure formation wasassessed. A 5% w/v SDP Solution and a 5% w/v Prior Art Silk FibroinSolution (100 μL each) were cast on 14 mm diameter silicone rubbersurfaces (n=6) and allowed to air dry into solid films over severalhours. The films were then assessed by ATR-FTIR (Nicolet iS10, ThermoScientific, MA) at a 4 nm resolution of 16 scans each.

The films were also processed for 5 hours within a water-annealingchamber, which is a vacuum container with water filled in the basin tocreate a 100% RH environment. The water vapor induces secondarystructure formation of fibroin protein films, most notably beta sheetstructures as shown by Jin et al. (Advanced Functional Materials 2005,15:1241-1247). The film samples were then reanalyzed with the FTIR asdescribed above. Spectral analysis revealed that SDP and Prior Art SilkFibroin films produced similar IR signatures before material processing,but the SDP material lacked the ability to form beta sheet secondarystructures post-processing as noted by the absence of well-known betasheet absorption peaks in the Amide I and II regions of the IR spectrumat 1624 cm⁻¹ and 1510 cm⁻¹, respectively (FIG. 9A). This findingrepresents a significant difference in material composition of the twosamples, which is a direct indication that the amino acid chemistry isinherently different in the SDP and PASF samples.

To represent the impact of secondary structure functionally, 150 mLsamples of the solutions were both dried within a convective clean airenvironment at room temperature for 48 hours. This resulted in theformation of solid protein material that demonstrated significantdifferences in appearance between the two solutions (FIG. 9B). Mostnotably, the autoclave-processed SDP material demonstrated a darkeryellow translucency that indicates chemical changes to aromatic aminoacids, when compared to the transparent and more pellucid PASF material.In addition, the SDP material formed a dried skin that prevented thelower region of the volume from completely dehydrating and thuspartially remained liquid. This was not the case for the Prior Art SilkFibroin material, which was completely dried and physically distortedinto a wavy material. These results indicate significant changes to thematerial's mechanical properties, and thus chemical interactions, as aresult of the autoclave processing to form the SDP material.

To assess solubility as a function of the autoclave processing, samplesof both dried materials were weighed and reconstituted in deionizedwater (diH₂O). For the SDP material the tough outer skin later waspeeled off and weighed, while for the Prior Art Silk Fibroin a portionof the material was broken off and weighed. For both samples, 500 mg ofmaterial was added to 25 mL of diH₂O (2% w/v solution) and then vortexedat high-speed setting for 10 minutes. Interestingly, the SDP materialcompletely dissolved in the diH₂O volume, while the Prior Art SilkFibroin material dissolved only very minimally (FIG. 9C). These resultsindicate the material solubility and solubility chemistry was distinctlychanged between the SDP and Prior Art Silk Fibroin materials due to theautoclave processing.

Example 9. Impact of Enzymatic Fibroin Cleavage on Solution Stability

To identify whether the increased stability of SDP is a directconsequence of amino acid transformation or merely due to the productionof smaller fibroin proteins generated by hydrolysis, Prior Art SilkFibroin (PASF) was treated with the serine protease trypsin toenzymatically break down fibroin as has been performed by Shaw (Biochem.J. 1964, 93(1), 45-54). In brief, 0.5-1.0 mg/mL of trypsin isolated frombovine pancreas (Sigma-Aldrich, T1426, MO) was added to PASF (78 mg/mL)solution containing HEPES buffer salts, mixed, and then incubated at 37°C. for 1, 2, 4, or 6 hours. Reactions were stopped with the addition of2 mM phenylmethylsulfonyl fluoride (PMSF), and the extent of fibroinfractionation was measured by 1D-PAGE and densitometry as described inExample 3.

TABLE 1 Average molecular weight of prior art silk fibroin enzymaticallycleaved with trypsin over increasing durations. Average MolecularTreatment Weight (kDa) PASF (Control) 107 1 hour trypsin 93 2 hourtrypsin 79 4 hour trypsin 70 6 hour trypsin 70

As shown in Table 1, trypsin treatment proved effective to progressivelyreduce the average molecular weight of PASF until 4 hours. Thesematerials were then subjected to ultrasonication to initiate beta-sheetformation and gelation as performed and described by Wang et al.(Biomaterials 2008. 29(8):1054-1064). The fractionation of PASF withtrypsin caused a dramatic acceleration in the kinetics of gelation,however, which is summarized in FIG. 10. Specifically, 1 hour trypsintreatment of PASF induced gel formation by ˜40 minutes followingsonication, which was slowed to ˜60 minutes in PASF exposed to trypsinfor 4 hours. Control PASF (in the presence of deactivated trypsin)exhibited increasing instability with time which reached maximalbeta-sheet formation (indicated by absorbance at 550 nm, FIG. 10) byapproximately 1300 minutes (data not shown). In contrast, silk-derivedproteins (SDP) showed no tendency toward instability during this timeframe, evidenced by a minimal and unchanging absorbance at 550 nm (FIG.10). These results indicate that fractionation of PASF by enzymaticcleavage of select peptide bonds, without amino acid transformation, areineffective and in fact counter-productive to forestall beta-sheetformation, instability, and gel formation.

Example 10. Impact of Disulfide Bonds on Fibroin Stability

The association between the fibroin heavy and light chain dimers existsthrough a single covalent disulfide bond, as elucidated by Tanaka et al.(Biochim Biophys Acta. 1999, 1432(1):92-103). Dimer separation caninstigate fibroin peptide-peptide interactions, which culminate ininsoluble protein aggregation (Shulha et al., Polymer 2006,47:5821-5830) that precedes beta-sheet formation and gelation (Nagarkaret al., Phys. Chem. Chem. Phys. 2010, 12:3834-3844). Therefore, todetermine whether disruption of the fibroin heavy-light chain dimerstimulates protein aggregation and therefore instability, PASF wastreated with the disulfide bond reducing agent dithiothreitol (DTT,Sigma-Aldrich, MO) at 0 (control), 10, or 100 mM, and was then subjectedto ultrasonication to instigate beta-sheet and gel formation. As shownin FIG. 11, the reduction of the disulfide bond in PASF with 10 mM DTTdecelerated, but did not inhibit instability, indicated by increasing550 nm absorbance over time relative to control samples. These effectswere further pronounced with 100 mM DTT, but still ineffective atforestalling instability. In contrast, SDP exhibited no tendency towardinstability following ultrasonication, indicated by an unchangingbaseline absorbance, which was unaffected by the addition of DTT (FIG.11). Collectively, these results demonstrate that the fibroin disulfidebridge participates in the mechanisms underlying PASF instability, buttheir reduction is ineffective to prohibit beta-sheet formation andgelation.

Example 11. Fibroin Stability Requires Heat in the Presence of LithiumBromide

To determine whether the heat-mediated transformation of amino acids inPASF requires lithium bromide, studies were undertaken to identify ifsimilar stability could be achieved when PASF was heated in the finalaqueous solution (lacking lithium bromide). To this end, PASF wasprepared (without additional heating) at 50 mg/mL concentration and thenheated in a jacketed reaction beaker (Chemglass, CG-1103-01, NJ)connected to a heater/chiller (Neslab, RTE-7, ThermoScientific, MA)actively circulating silicone oil heat exchange fluid (AceGlass,14115-05, NJ). The circulator was set to ˜200° F., the temperature justbelow the boiling point of PASF lacking lithium bromide salts, and wasallowed to stabilize for 15±1 minutes. PASF (25 mL) was incubated in thereaction beaker with a PTFE-coated stir bar and placed on a stir plate(IKA C-MAG HS7, NC) to ensure solution temperature homogeneity. PASFtemperature was actively monitored throughout the heating period usingan external thermocouple (Omega HH-603, Omega Engineering, CT), andsamples (3 mL) were removed at the following timepoints:

Temperature Timepoint  68° F.  0 min 196° F.  30 min 196° F.  60 min196° F.  90 min 196° F. 120 min

Drawn samples were then subjected to ultrasonication to instigatebeta-sheet formation and gelation, as has been described previously byWang et al. (Biomaterials 2008, 29(8):1054-1064). To compare theseresults with PASF that had been heated in the presence of lithiumbromide, SDP was also ultrasonicated separately, and absorbance at 550nm monitored longitudinally to compare the kinetics of fibroininstability. As shown in FIG. 12, the application of heat to PASFactually increased baseline absorbance and hence instability relative tonon-heated control samples. Furthermore, the duration of heat exposure(from 30 to 120 minutes) to PASF was inversely proportional to basalabsorbance, indicating that the presence of lithium bromide duringproduction of SDP is needed to achieve the minimal absorbance observedin this latter solution (FIG. 12). Furthermore, 550 nm absorbancecontinued to escalate in all of the heated PASF solutions over time butdid not change from baseline in ultrasonicated SDP solutions, thusclearly demonstrating that heat-treated samples were undergoingbeta-sheet formation and therefore becoming unstable. Collectively,these results indicate that heat mediated hydrolysis of PASF in theabsence of lithium bromide is insufficient to mediate the amino acidtransformations that facilitate protein stability to the same degree asfor the SDP described herein.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents cited herein areincorporated by reference herein, as though individually incorporated byreference. No limitations inconsistent with this disclosure are to beunderstood therefrom. The invention has been described with reference tovarious specific and preferred embodiments and techniques. However, itshould be understood that many variations and modifications may be madewhile remaining within the spirit and scope of the invention.

1. A fibroin-derived protein composition prepared by a processcomprising heating an aqueous fibroin solution at an elevated pressure,wherein: the aqueous fibroin solution comprises lithium bromide at aconcentration of at least about 8M, and the aqueous fibroin solution isheated to at least about 105° C. (221° F.) under a pressure of about 10PSI to about 20 PSI for at least about 20 minutes in the presence of thelithium bromide; to provide a protein composition comprisingfibroin-derived protein wherein: the primary amino acid sequences of thefibroin-derived protein composition differ from native fibroin by atleast by at least 4% with respect to the combined amino acid content ofserine, glycine, and alanine, based on the combined absolute values ofthe differences in the content of serine, glycine, and alanine; cysteinedisulfide bonds between the fibroin heavy and fibroin light proteinchains of fibroin are reduced or eliminated; the protein composition hasa serine content that is reduced by greater than 25% compared to nativefibroin protein; and wherein the average molecular weight of thefibroin-derived protein composition is less than about 100 kDa.
 2. Theprotein composition of claim 1 wherein the aqueous fibroin solution isheated to a temperature of about 115° C. (239° F.) to about 125° C.(257° F.) under a pressure of about 15 PSI to about 20 PSI for at leastabout 30 minutes.
 3. The protein composition of claim 1 wherein thefibroin-derived protein has an average molecular weight of less thanabout 60 kDa.
 4. The protein composition of claim 1 wherein the proteincomposition has an aqueous viscosity of less than 5 cP as a 10% w/wsolution in water.
 5. The protein composition of claim 1 wherein theprotein composition has an aqueous viscosity of less than 10 cP as a 24%w/w solution in water.
 6. The protein composition of claim 1 wherein thefibroin-derived protein comprises between 4.5% and 7.5% serine aminoacid residues.
 7. The protein composition of claim 1 wherein a primaryamino acid sequences of the fibroin-derived protein differ from nativefibroin by at least 8% with respect to the combined amino acid contentof serine, glycine, and alanine based on a combined absolute values ofthe differences in the content of serine, glycine, and alanine.
 8. Theprotein composition of claim 1 wherein the fibroin-derived proteincomprises less than 8.5% serine amino acid residues.
 9. The proteincomposition of claim 8 wherein the fibroin-derived protein comprisesless than 6% serine amino acid residues.
 10. The protein composition ofclaim 1 wherein the fibroin-derived protein comprises greater than 46.5%glycine amino acids.
 11. The protein composition of claim 10 wherein thefibroin-derived protein comprises greater than 48% glycine amino acids.12. The protein composition of claim 1 wherein the fibroin-derivedprotein comprises greater than 30% alanine amino acids.
 13. The proteincomposition of claim 12 wherein the fibroin-derived protein comprisesgreater than 31.5% alanine amino acids.
 14. A method of treating dry eyesyndrome in a subject comprising administering an effective amount ofthe protein composition of claim 1 to an eye of the subject, therebytreating the dry eye disease, wherein the protein composition isformulated as an ophthalmic formulation comprising water.
 15. The methodof claim 14 wherein the protein composition comprises about 0.1% toabout 10% by weight of the ophthalmic formulation.
 16. The method ofclaim 15 wherein the protein composition comprises one or more of abuffering agents, a stabilizing agents, and a demulcent.
 17. The methodof claim 16 wherein the demulcent is one or more of hyaluronic acid(HA), hydroxyethyl cellulose, hydroxypropyl methylcellulose, dextran,gelatin, a polyol, carboxymethyl cellulose (CMC), polyethylene glycol,propylene glycol (PG), hypromellose, glycerin, polysorbate 80, polyvinylalcohol, and povidone, and wherein the buffering agent is one or more ofphosphate buffered saline, borate buffered saline, citrate buffersaline, sodium chloride, calcium chloride, magnesium chloride, potassiumchloride, sodium bicarbonate, zinc chloride, hydrochloric acid, sodiumhydroxide, and edetate disodium.